The recommended dietary allowance for vitamin C (ascorbate) is based on preventing the deficiency disease scurvy. We proposed that a new recommended dietary allowance for vitamin C requirements can be determined using the concept of in situ kinetics. In situ kinetics has biochemical and clinical components. The goal of the biochemical component is to determine vitamin C molecular function in relation to its concentration. For these studies, vitamin is investigated in human fibroblasts, lymphocytes, and neutrophils. To determine how intracellular concentration is regulated, vitamin transport has been characterized. Ascorbate transport is concentration dependent, saturable mediated by a sodium-dependent carrier, and inhibited by newly synthesized vitamin C analogs. Oxidized ascorbate dehydroascorbic acid is transported separately by GLUT I and immediately reduced intracellularly to ascorbate. Several proteins have been isolated which mediate dehydroascorbic acid reduction to ascorbate in neutrophils. One protein was responsible for the majority of reducing activity and was isolated, sequenced, identified as thioltransferese (glutaredoxin), cloned, and modified by site-directed mutagenesis. Cloning of genomic glutaredoxin and characterization of the promoter region are underway. These studies indicate that vitamin C function in neutrophils may be to protect neutrophils from their own oxidants. Overall findings for fibroblasts, neutrophils, and lymphocytes suggest that kinetics for vitamin C function in situ can be determined in relationship to vitamin concentration in situ. The clinical component of in situ kinetics is to determine how vitamin concentrations are achieved in normal humans as a function of dose and whether concentrations humans achieve are those required for molecular functions of the vitamin. A clinical trial completed in healthy men invested the relationship between vitamin C dose and its concentration in plasma and tissues, bioavailability, urinary excretion, and potential adverse effects. A clinical trial in women is underway. Based on biochemical and clinical in situ kinetics, we are recommending 200mg as a new recommended dietary allowance for vitamin C in healthy men.